The Human FETU-B ELISA Kit is designed for ease of use and does not require additional reagents or equipment beyond what is provided in the kit. The following items are needed:
1. A microplate reader capable of measuring at 450 nm.
2. A high-precision pipette with tips suitable for volumes of 0.5–10 µL, 2–20 µL, 20–200 µL, and 200–1000 µL.
3. A 37°C incubator.
4. Distilled or deionized water.
**Notes:**
After testing a large number of normal samples, it was found that most specimens fall within the detection range of the kit. Therefore, you can directly use 50 µL of the sample during the experiment. However, if some sample values exceed the maximum standard concentration, the sample should be diluted with the provided sample diluent before proceeding.
**Precautions:**
1. Strictly follow the specified incubation time and temperature to ensure accurate results. All reagents must reach room temperature (20–25°C) before use. Store unused reagents in the refrigerator immediately after use.
2. Inadequate washing may lead to inaccurate readings. Make sure to remove as much liquid as possible from each well before adding the substrate. Keep the microplates moist throughout the incubation process.
3. Remove any residual liquid and fingerprints from the bottom of the plate, as they may affect the OD reading.
4. The substrate solution should be colorless or very light. If it turns blue, it should not be used.
5. Avoid cross-contamination between reagents and samples to prevent erroneous results.
6. Protect the kit from direct exposure to strong light during storage and incubation.
7. After equilibrating to room temperature, open the sealed bag carefully to avoid condensation on the microplate.
8. Do not expose any reagents to strong gases from bleach or bleaching agents, as they may damage the biological activity of the reagents.
9. Never use expired kits.
10. If there is a possibility of disease transmission, all samples should be handled according to proper biosafety protocols. The reagent kit should be brought to room temperature before use.
**Reagent Preparation:**
Dilute the 20× Wash Buffer by mixing 1 part of the buffer with 19 parts of distilled water.
**Procedure:**
1. Remove the required microplate strips from the foil pouch after allowing them to equilibrate at room temperature for 20 minutes. Store the remaining strips in a ziplock bag in the refrigerator at 4°C.
2. Set up the standard and sample wells. Add 50 µL of each standard solution to the corresponding wells.
3. Add 50 µL of the sample to be tested into the sample wells. Do not add anything to the blank wells.
4. Add 100 µL of HRP-labeled detection antibody to each well (except the blank), cover with a sealing film, and incubate at 37°C for 60 minutes.
5. Discard the liquid, blot dry with absorbent paper, then add 350 µL of wash buffer to each well. Let stand for 1 minute, discard the buffer, blot dry, and repeat this washing process 5 times (or use an automated washer if available).
6. Add 50 µL of substrate A and B to each well, and incubate in the dark at 37°C for 15 minutes.
7. Add 50 µL of stop solution to each well, and measure the OD value at 450 nm within 15 minutes.
To calculate the results, plot the OD values of the standards against their concentrations on a graph or using software. Determine the linear regression equation and use it to calculate the concentration of the unknown samples based on their OD values.
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